Gas chromatography-mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking.

RATIONALE: Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). METHODS: Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 µL was performed using GC-MS, and selected ion monitoring (SIM) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT. RESULTS: The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT. CONCLUSIONS: A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC-MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of samples on a daily routine basis.

Venous thromboembolism at uncommon sites in neonates and children.

We retrospectively analyzed the data of 24 children (whereof 11 neonates), with non-central venous line-related and nonmalignancy-related venous thromboembolism (VTE) at uncommon sites, referred to our Unit from January 1999 to January 2012. Thirty patients who also suffered deep vein thrombosis, but in upper/low extremities, were not included in the analysis. The location of rare site VTE was: portal (n=7), mesenteric (n=2) and left facial vein (n=1), spleen (n=3), lung (n=3), whereas 10 neonates developed renal venous thrombosis. The majority of patients (91.7%) had at least 1 risk factor for thrombosis. Identified thrombophilic factors were: antiphospholipid antibodies (n=2), FV Leiden heterozygosity (n=6), MTHFR C677T homozygosity (n=4), protein S deficiency (n=2), whereas all neonates had age-related low levels of protein C and protein S. All but 6 patients received low-molecular-weight heparin, followed by warfarin in 55% of cases, for 3 to 6 months. Prolonged anticoagulation was applied in selected cases. During a median follow-up period of 6 years, the clinical outcome was: full recovery in 15 patients, evolution to both chronic portal hypertension and esophageal varices in 2 children, and progression to renal failure in 7 of 10 neonates. Neonates are greatly vulnerable to complications after VTE at uncommon sites, particularly renal. Future multicentre long-term studies on neonatal and pediatric VTE at unusual sites are considered worthwhile.

Assessment of bone mineral density and markers of bone turnover in children under long-term oral anticoagulant therapy.

Oral anticoagulants antagonize vitamin K action and potentially impair the carboxylation of osteocalcin, a protein essential for normal bone matrix formation. In the present study, bone mineral density (BMD) and bone turnover markers were evaluated in 23 children under long-term oral anticoagulant therapy. BMD of the lumbar spine was assessed (Dual Energy x-ray Absorptiometry) and reported as z score. Osteoblast [bone alkaline phosphatase, osteocalcin (Gla-Oc), amino-terminal procollagen 1 extension peptide] and osteoclast (urinary calcium and deoxypyridinoline, serum cross-linked C telopeptide) activity markers were measured. Vitamin D {[25(OH) D], parathormone, calcium, phosphorus, magnesium} and vitamin K status [factors II, VII, IX, X, protein C, protein S, undercarboxylated osteocalcin (Glu-Oc)] were determined. The above parameters were also evaluated in 25 healthy controls. Patients presented with higher levels in Glu-Oc, parathormone, and bone resorption markers, lower levels in bone formation markers and 25(OH) D, whereas 52% of them showed signs of osteopenia (-1>BMD z score>-2.5). Statistical analysis demonstrated that anticoagulant therapy was an independent predictor of alterations in Glu-Oc, Gla-Oc, bone alkaline phosphatase, amino-terminal procollagen 1 extension peptide, and serum cross-linked C telopeptide levels. It seems that long-term use of coumarin derivatives may cause osteopenia in children with the risk of developing osteoporosis later in life.